Vijaya Iragavarapu-Charyulu

Identification of a Subpopulation of Macrophages in Mammary Tumor–Bearing Mice That Are Neither M1 nor M2 and Are Less Differentiated

Systemic and local immune deficiency is associated with cancer, and the role of M2 tumor-associated macrophages in this phenomenon is well recognized. However, the immune status of macrophages from peripheral compartments in tumor hosts is unclear. Peritoneal macrophages (PEM) are derived from circulating monocytes and recruited to the peritoneal cavity where they differentiate into macrophages. We have previously shown that PEMs from mice bearing D1-DMBA-3 mammary tumors (T-PEM) are deficient in inflammatory functions and that this impairment is associated with diminished expression of transcription factors nuclear factor kappaB and CAAT/enhancer-binding protein. We now provide evidence that T-PEMs display neither M1 nor M2 phenotypes, yet exhibit deficiencies in the expression of several inflammatory cytokines and various proinflammatory signaling pathways. Moreover, due to nuclear factor kappaB down-regulation, increased apoptosis was observed in T-PEMs. We report for the first time that macrophage depletion is associated with increased macrophage progenitors in bone marrow. Furthermore, T-PEMs have a lower expression of macrophage differentiation markers F4/80, CD68, CD115, and CD11b, whereas Gr-1 is up-regulated. Our results suggest that T-PEMs are less differentiated and represent a newly derived population from blood monocytes. Lastly, we show that transforming growth factor-beta and prostaglandin E(2), two immunosuppressive tumor-derived factors, may be involved in this phenomenon.

Up-Regulation of Matrix Metalloproteinase-9 in T Lymphocytes of Mammary Tumor Bearers: Role of Vascular Endothelial Growth Factor

Matrix metalloproteinase-9 (MMP-9), a matrix-degrading enzyme, is crucial in tumor invasion and metastasis and is implicated in leukocyte extravasation. In this report, we demonstrate that during growth of the D1–7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is progressive up-regulation of MMP-9 in splenic T cells at both the transcriptional and translational levels. Our previous work has identified several factors produced by this tumor, including PGE2, GM-CSF, and phosphatidyl serine; however, none of these agents induces increased production of MMP-9 by normal splenic T cells. Although not produced by the tumor, TNF-α and IL-6 are up-regulated in both macrophages and B cells in tumor-bearing mice. Exposure of normal T cells to these two cytokines, however, also fails to up-regulate MMP-9 production. Vascular endothelial growth factor (VEGF) is produced by many tumors, and we determined that the mammary tumor used in our studies expresses high levels of this angiogenic growth factor. Importantly, splenic T cells from tumor bearers constitutively produce increased amounts of VEGF, and treatment of normal T cells with VEGF results in up-regulated MMP-9 production. Of crucial importance is the finding that tumor-infiltrating T cells also produce high levels of VEGF and MMP-9. Our studies indicate that VEGF can act directly on T lymphocytes and that elevated VEGF levels may contribute to the aberrant MMP-9 secretion by mammary tumor bearers’ T cells.

Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model

Disseminated metastasis accounts for over 90% of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase‐3 like‐protein‐1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. In this study, we show that there are increased levels of CHI3L1 in plasma of tumor‐bearing mice and that both tumor cells and immune cells express and secrete CHI3L1. However, the biological and physiological functions of CHI3L1 are still unclear. We demonstrate that while CHI3L1 has an inhibitory role in the expression of interferon‐gamma (IFN‐γ), CHI3L1 up‐regulates pro‐inflammatory mediators, C‐chemokine ligand 2 (CCL2), chemokine CX motif ligand 2 (CXCL2) and matrix metalloproteinase‐9 (MMP‐9) all of which contribute to tumor growth and metastasis. We found that in vitro inhibition of CHI3L1 by siRNA suppressed the production of CCL2, CXCL2 and MMP‐9 by macrophages. In vivo treatment of mammary tumor‐bearing mice with chitin (β‐(1‐4)‐poly‐N‐acetyl D‐glucosamine), a TH1 adjuvant and a ligand for CHI3L1, promoted immune effector functions with increased production of IFN‐γ and decreased CCL2, CXCL2 and MMP‐9 expression. In vivo administration of chitin to mammary tumor‐bearing mice significantly decreased lung metastasis. These studies show that CHI3L1 plays a role in tumor progression and that chitin can inhibit the pleiotropic effects of CHI3L1 giving support to the idea that CHI3L1 is a useful therapeutic target for treatment of breast cancer.

CHI3L1 plays a role in cancer through enhanced production of pro-inflammatory/pro-tumorigenic and angiogenic factors

Elevated serum levels of a glycoprotein known as chitinase-3-like protein 1 (CHI3L1) have been correlated with poor prognosis and shorter survival of patients with cancer and inflammatory diseases. The biological and physiological functions of CHI3L1 in cancer have not yet been completely elucidated. In this review, we describe the role of CHI3L1 in inducing pro-inflammatory/pro-tumorigenic and angiogenic factors that could promote tumor growth and metastasis.

Tumor microenvironment profoundly modifies functional status of macrophages: Peritoneal and tumor-associated macrophages are two very different subpopulations

Macrophages are key players in the inflammatory response. In this study, we tested the hypothesis that although all macrophage subpopulations in tumor hosts are affected by the disease, it is the close proximity to the tumor that induces major alterations in these cells. We compared tumor-associated macrophages (TAMs) with peritoneal macrophages from mice bearing D1-DMBA-3 mammary tumors (T-PEMs). Our results show that TAMs downregulate IL-12p70 but upregulate IL-12p40, IL-23, IL-6 and IL-10. Some NFκB and C/EBP transcription factors family members are decreased in TAMs; however NFκBp50 homodimers, STAT1/pSTAT1 and STAT3/pSTAT3 are overexpressed. Furthermore, while TAMs block T-cell proliferation and are more prone to apoptosis compared to T-PEMs, both types of macrophages have an impaired phagocytic capacity. Moreover, TAMs constitutively express iNOS and produce nitric oxide but do not express arginase and are Gr-1(high) and CD11b(low). Collectively, our analysis of two spatially distinct macrophage subpopulations in tumor-bearing mice revealed that the tumor modulates them differently into two molecularly and functionally dissimilar macrophage subpopulations.

Semaphorin7A promotes tumor growth and exerts a pro-angiogenic effect in macrophages of mammary tumor-bearing mice

Semaphorins are a large family of molecules involved in axonal guidance during the development of the nervous system and have been recently shown to have both angiogenic and anti-angiogenic properties. Specifically, semaphorin 7A (SEMA7A) has been reported to have a chemotactic activity in neurogenesis and to be an immune modulator through α1β1integrins. SEMA7A has been shown to promote monocyte chemotaxis and induce them to produce proinflammatory mediators. In this study we explored the role of SEMA7A in a murine model of breast cancer. We show that SEMA7A is highly expressed by DA-3 murine mammary tumor cells in comparison to normal mammary cells (EpH4), and that peritoneal elicited macrophages from mammary tumor-bearing mice also express SEMA7A at higher levels compared to those derived from normal mice. We also show that murine macrophages treated with recombinant murine SEMA7A significantly increased their expression of proangiogenic molecule CXCL2/MIP-2. Gene silencing of SEMA7A in peritoneal elicited macrophages from DA-3 tumor-bearing mice resulted in decreased CXCL2/MIP-2 expression. Mice implanted with SEMA7A silenced tumor cells showed decreased angiogenesis in the tumors compared to the wild type tumors. Furthermore, peritoneal elicited macrophages from mice bearing SEMA7A-silenced tumors produce significantly (p < 0.01) lower levels of angiogenic proteins, such as CXCL2/MIP-2, CXCL1, and MMP-9, compared to those from control DA-3 mammary tumors. We postulate that SEMA7A in mammary carcinomas may skew monocytes into a pro-tumorigenic phenotype to support tumor growth. SEMA7A could prove to be valuable in establishing new research avenues toward unraveling important tumor-host immune interactions in breast cancer patients.

YKL-40/CHI3L1 drives inflammation on the road of tumor progression

Inflammation plays a vital role at different stages of tumor progression. The development of tumors is affected by inflammatory mediators produced by the tumor and the host. YKL‐40/chitinase‐3‐like‐1 protein is often up‐regulated in inflammation‐associated diseases. With the use of chronic inflammatory disease systems, we describe the role of YKL‐40/chitinase‐3‐like‐1 protein in enhancing the inflammatory response and its implications in tumorigenesis. We also discuss how pre‐existing inflammation enhances tumor growth and metastasis. In this mini‐review, we highlight the effect of YKL‐40/chitinase‐3‐like‐1 protein‐associated inflammation in promoting tumor progression.

Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1–3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice☆

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.

Exploring the role of CHI3L1 in “pre-metastatic” lungs of mammary tumor-bearing mice

Elevated levels of chitinase-3-like-1 (CHI3L1) are associated with poor prognosis, shorter recurrence-free intervals and low survival in breast cancer patients. Breast cancer often metastasizes to the lung. We hypothesized that molecules expressed in the “pre-metastatic” lung microenvironment could support the newly immigrant tumor cells by providing growth and angiogenic factors. Macrophages are known to play an important role in tumor growth by releasing pro-angiogenic molecules. Using mouse mammary tumor models, we have previously shown that during neoplastic progression both the mammary tumor cells and splenic macrophages from tumor-bearing mice express higher levels of CHI3L1 compared to normal control mice. However, the role of CHI3L1 in inducing angiogenesis by macrophages at the pulmonary microenvironment to support newly arriving breast cancer cells is not yet known. In this study, we determined the expression of CHI3L1 in bronchoalveolar lavage macrophages and interstitial macrophages in regulating angiogenesis that could support the growth of newly immigrant mammary tumor cells into the lung. Here we show that in vitro treatment of pulmonary macrophages with recombinant murine CHI3L1 resulted in enhanced expression of pro-angiogenic molecules including CCL2, CXCL2, and MMP-9. We and others have previously shown that inhibition of CHI3L1 decreases the production of angiogenic molecules. In this study, we explored if in vivo administration of chitin microparticles has an effect on the expression of CHI3L1 and pro-angiogenic molecules in the lungs of mammary tumor-bearing mice. We show that treatment with chitin microparticles decreases the expression of CHI3L1 and pro-angiogenic molecules in the “metastatic” lung. These studies suggest that targeting CHI3L1 may serve as a potential therapeutic agent to inhibit angiogenesis and thus possibly tumor growth and metastasis.

The regulation of angiogenesis by tissue cell-macrophage interactions.

Angiogenesis, the sprouting of new blood vessels from existing ones, is a process important both in physiological and pathogenic conditions. During angiogenesis, endothelial cells proliferate, migrate and organize into new, functional blood vessels. Macrophages play an important role in this process as they process microenvironmental cues, and directly secrete or stimulate other cell types to secrete pro-angiogenic mediators, including chemokines, cytokines and growth factors that together stimulate endothelial cell proliferation, degrade the extracellular matrix (ECM), and attract leukocytes to further enhance angiogenesis. Since macrophages are extremely plastic and can be differently activated by various stimuli, they exert many regulatory effects on angiogenesis, although many aspects of this regulation are still unclear. This volume highlights several examples of macrophages-driven regulation of angiogenesis. We begin with an introductory review which describes the role of tumor-associated macrophages (TAMs) in angiogenesis and lymphangiogenesis. Riabov et al. (2014) describe the crosstalk between tumor cells and TAMs in hypoxic and cytokine-rich microenvironment, resulting in the induction of pro-angiogenic behavior in both cell types. We then chose to highlight the role of the chitinases, a family of proteins with newly discovered roles in angiogenesis. In this review by Shao (2013), the roles and mode of action of YKL-40/chitinase-3-like-1 (CHI3L1) are described focusing on its angiogenic signature pertaining to tumor vascularization and development. YKL-40 regulates tumor vascularization mediated by endothelial cells and promotes vascular integrity supported by smooth muscle cells. Shao also reports that while YKL-40-induced angiogenic response in endothelial cell is VEGF-independent, YKL-40 itself is induced when VEGF is inhibited. Next, an original paper by Libreros et al. (2013) suggests that increased levels of CHI3L1 produced by pulmonary macrophages set up the pre-metastatic niche in breast cancer. CHI3L1 enhances angiogenesis by inducing chemokines (CCL2 and CXCL2) and MMP-9 expression, and attraction of macrophages into the lung. The importance of CHI3L1 as a regulator of angiogenesis, as well as possible target of treatment, is demonstrated by the addition of chitin microparticles that can inhibit CHI3L1, chemokines and MMP-9 production in the pre-metastatic lung. Several pro-inflammatory mediators are long known to be involved in angiogenesis, and the next three reviews elaborate on these. Owen and Mohamadzadeh (2013) describe the potential roles of M2 TAMs in modulating angiogenesis through production of chemokines. The role of the pro-angiogenic ELR+ chemokines that attract neutrophils, and the angiostatic ELR-chemokines, with the exception of CXCL12, are discussed in the context of tumor progression. M1 vs. M2 secreted profiles of cytokines/chemokines which may play roles in immune regulation, angiogenesis, tumor progression and metastasis are highlighted. Voronov et al. (2014) describe the roles of the IL-1 family of proteins in angiogenesis, and especially IL-1β that can directly drive endothelial cells to proliferate and generate tube-like structures, and to secrete pro-angiogenic cytokines and chemokines. Additionally, IL-1α attracts macrophages and collaborates with VEGF to enhance angiogenesis. Knockout mice for each of the different members of the family help reveal that IL-1β, and to a lesser extent IL-1α, are responsible for the pro-angiogenic effects, and therefore IL-1β depletion could reduce angiogenesis. The dual role of nitric oxide in tumor biology, as a cytotoxic factor for tumor cells, and as a pro-angiogenic factor, is summarized by Rahat and Hemmerlein (2013). The authors caution against the common use of iNOS immunohistochemical staining as a prognostic factor, as no correlation between survival rates, invasiveness or tumor recurrence after therapy is found. This is due to the ability of the microenvironment (e.g. hypoxia) to inhibit iNOS activity, even when it is highly expressed. Furthermore, tumor cells can manipulate their own expression of iNOS through the expression of microRNA-146a, to control their fate and evade macrophage-induced cell death, suggesting new possible therapeutic approaches. Two original papers demonstrate the pro-angiogenic role of additional, less studied proteins. Garcia-Areas et al. (2014) suggest that semphorin 7A (SEMA7A), a member of the semaphorin family involved in neuronal axonal guidance, is also a pro-angiogenic factor whose expression is increased in tumor-bearing mice. When triggered by SEMA7A, macrophages elevate secretion of angiogenic CXCL2/MIP-2 while silencing SEMA7A resulted in decreased tumor angiogenesis, and lower levels of CXCL2/MIP-2, CXCL1 and MMP-9. Amit-Cohen et al. (2013) report that EMMPRIN, a transmembranal protein that is overexpressed in tumor cells and have a pro-angiogenic activity, can induce the expression of MMP-9 and VEGF from macrophages in a secreted form instead of membranal form. Tumor cells and macrophages must be co-cultured for this effect, emphasizing the importance of their interaction. Secreted EMMPRIN levels were elevated through shedding-off of the membranal protein by the activity of a serine protease that was not yet identified. The final paper sheds light on the clinical aspect of vasculature re-growth after radiation therapy. Russell and Brown (2013) argue that because radiation destroys endothelial cells and tumor vasculature, tumor hypoxia is markedly increased resulting in induction of HIF-1 and HIF-2. These factors enhance the secretion of pro-angiogenic cytokines, chemokines and growth factors and recruit bone marrow-derived monocytes and macrophages which differentiate into TAMs and TEMs in the tumor. Thus, targeting the infiltration process after radiation therapy may improve the efficiency of treatment, prevent re-establishment of new vasculature and reduce tumor recurrence. Collectively, the articles in this topic highlight the importance and complexity of the interactions between macrophages and tumor cells in angiogenesis. Macrophages emerge as key regulators of the process because of their ability to communicate with tumor or stromal cells, sense the microenvironment and respond by inducing secretion of potent pro-angiogenic mediators. Some of these pro-angiogenic mediators and the triggers for their enhanced expression and secretion are at the focus of the current topic. Although more research is required, it is clear that tumor cell-macrophage interactions must precede and regulate the secretion phase—a point that was emphasized throughout this volume. This regulatory ability of the macrophages, and the fact that many mediators are both pro-inflammatory and pro-angiogenic, strongly links inflammation and angiogenesis together. Many inflammatory diseases, including cancer, are characterized by enhanced angiogenesis and a prominent macrophage component. The findings described here may be relevant for understanding macrophage-angiogenesis networks and potential therapy designs.